Throughout this specification, including any claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and any appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Methylthioninium Chloride (MTC) (Also Known as Methylene Blue)
Methylthioninium Chloride (MTC) (also known as Methylene Blue (MB); methylthionine chloride; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathionium chloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet; and Urolene Blue®) is a low molecular weight (319.86), water soluble, tricyclic organic compound of the following formula:

Methylthioninium Chloride (MTC) (also known as Methylene Blue), perhaps the most well-known phenothiazine dye and redox indicator, has also been used as an optical probe of biophysical systems, as an intercalator in nanoporous materials, as a redox mediator, and in photoelectrochomic imaging.
See, for example, Colour Index (Vol. 4, 3rd edition, 1971) and Lillie et al., 1979, and references cited therein.
MTC is currently used to treat methemoglobinemia (a condition that occurs when the blood cannot deliver oxygen where it is needed in the body). MTC is also used as a medical dye (for example, to stain certain parts of the body before or during surgery); a diagnostic (for example, as an indicator dye to detect certain compounds present in urine); a mild urinary antiseptic; a stimulant to mucous surfaces; a treatment and preventative for kidney stones; and in the diagnosis and treatment of melanoma.
MTC has been used to treat malaria either singly (Guttmann & Ehrlich, 1891) or in combination with chloroquine (Schirmer et al. 2003; Rengelhausen et al. 2004). Malaria in humans is caused by one of four protozoan species of the genus Plasmodium: P. falciparum, P. vivax, P. ovale, or P. malariae. All species are transmitted by the bite of an infected female Anopheles mosquito. Occasionally, transmission occurs by blood transfusion, organ transplantation, needle-sharing, or congenitally from mother to fetus. Malaria causes 300-500 million infections worldwide and approximately 1 million deaths annually. Drug resistance, however is a major concern and is greatest for P. falciparum, the species that accounts for almost all malaria-related deaths. Drugs or drug combinations that are currently recommended for prophylaxis of malaria include chloroquine/proguanil hydrochloride, mefloquine, doxycycline and primaquine.
MTC (under the name Virostat, from Bioenvision Inc., New York) has shown potent viricidal activity in vitro. Specifically Virostat is effective against viruses such as HIV and West Nile Virus in laboratory tests. West Nile virus (WNV) is a potentially serious illness affecting the central nervous system. The large majority of infected people will show no visible symptoms or mild flu-like symptoms such as fever and headache. About one in 150 will develop severe symptoms including tremors, convulsions, muscle weakness, vision loss, numbness, paralysis or coma. Generally, WNV is spread by the bite of an infected mosquito, but can also spread through blood transfusions, organ transplants, breastfeeding or during pregnancy from mother to child. Virostat is also currently in clinical trials for the treatment of chronic Hepatitis C. Hepatitis C is a viral infection of the liver. The virus, HCV, is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. HCV is spread primarily by direct contact with human blood. The major causes of HCV infection worldwide are use of unscreened blood transfusions, and re-use of needles and syringes that have not been adequately sterilized. The World Health Organization has declared hepatitis C a global health problem, with approximately 3% of the world's population infected with HCV and it varies considerably by region. The prevalence in the US is estimated at 1.3% or approximately 3.5 million people. Egypt contains the highest prevalence of hepatitis C in the world, estimated at over 20% of the nation's approximately 62 million people.
MTC, when combined with light, can prevent the replication of nucleic acid (DNA or RNA). Plasma, platelets and red blood cells do not contain nuclear DNA or RNA. When MTC is introduced into the blood components, it crosses bacterial cell walls or viral membrane then moves into the interior of the nucleic acid structure. When activated with light, the compounds then bind to the nucleic acid of the viral or bacterial pathogen, preventing replication of the DNA or RNA. Because MTC is designed to inactivate pathogens, it has the potential to reduce the risk of transmission of pathogens that would remain undetected by testing.
MTC and derivatives thereof (e.g., “diaminophenothiazinium compounds”) have been found to be useful in the treatment of tauopathies (such as, for example, Alzheimer's disease) (see, for example, Wischik, C. M., et al., 1996, 2002).
MTC was first described in a German Patent in 1877 (Badische Anilin- und Soda-Fabrik, 1877). In that patent, MTC was synthesized by nitrosylation of dimethylaniline, subsequent reduction to form N,N-dimethyl-1,4-diaminobenzene, and subsequent oxidative coupling in the presence of hydrogen sulphide (H2S) and iron(III) chloride (FeCl3).
Bernthsen described subsequent studies of MTC and methods for its synthesis (see Bernthsen, 1885a, 1885b, 1889).
Fierz-David and Blangley, 1949, also describes methods for the synthesis of MTC from dimethylaniline, as illustrated in the following scheme

In step (a), nitrosodimethylaniline is prepared from dimethylaniline by treatment with nitrite (NaNO2) in aqueous acid (HCl) solution. In step (b), the nitroso compound is reduced to form p-aminodimethylaniline using additional aqueous acid (HCl) solution using zinc dust. The metal residue after step (b) is removed by filtration and the filtrate is oxidised in the presence of thiosulfonic acid, sulphuric acid and non-reducing zinc chloride solution, step (c). Further oxidation in the presence of dimethylaniline results in the thiosulfonic acid of Bindschedlers green, step (d). The ring is then closed using manganese dioxide or copper sulphate to form methylene blue. More specifically, a clear neutral solution of p-aminodimethylaniline is acidified (H2SO4), and a non reducing zinc chloride solution is added (ZnCl2 with Na2Cr2O7). Aqueous aluminium sulphate (Al2(SO4)) and crystalline sodium thiosulphate (Na2S2O3) are added. Aqueous sodium dichromate (Na2Cr2O7) is added. The mixture is heated by dry steam. Aqueous acidic (HCl) dimethylaniline is then added. Aqueous sodium dichromate (Na2Cr2O7) is added. The mixture is heated with dry steam, and becomes dark greenish-blue in colour due to the formation of the thiosulfonic acid of Bindschedler green. An aqueous slurry of manganese dioxide or copper sulfate is added, and the mixture heated by dry steam, and the dye precipitates from the concentrated zinc chloride solution. To recover the dye from the mixture it is cooled and acidified (H2SO4) to dissolve the aluminium, manganese and chromium salts. The mixture is cooled further and the crude dye collected by filtration. Purification from water, sodium chloride and zinc chloride gives the zinc double salt of methylene blue as bronzy red crystals.
Very similar synthesis methods are described in the Colour Index (Vol. 4, 3rd edition, 1971), p. 4470.
Masuya et al., 1992, describe certain phenothiazine derivatives, and methods for their preparation and use in photodynamic therapy of cancer and in immunoassays utilizing chemiluminescence. The compounds are prepared by routes similar to those discussed above.
Leventis et al., 1997, describe methods for the synthesis of certain MTC analogs, which employ phenothiazine as a starting material and which add the desired 3,7-substituents by halogenation followed by amination. The authors assert that MTC is synthesized commercially by oxidation of N,N-dimethyl-p-phenylene diamine with Na2Cr2O7 in the presence of Na2S2O3, followed by further oxidation in the presence of N,N-dimethylamine.
Fierz-David et al., 1949, describes the synthesis of the zinc chloride double salt of MTC and the removal of zinc by chelation with sodium carbonate followed by filtration to generate zinc free methylene blue. However, the authors acknowledge that this technique cannot be used on a large scale, because the yields are poor.
Methods for synthesizing high purity MTC and its derivatives have been proposed in WO 2006/032879. The compounds are synthesized according to the scheme shown in FIG. 1.
These steps can be summarised as follows: (i) nitrosylation (NOS), (ii) nitrosyl reduction (NR), (iii) thiosulfonic acid formation (TSAF), (iv) oxidative coupling (OC), (v) ring closure (RC), and recrystallization (RX). A variant is also described, in which the thiosulfonic acid of Bindshedler's Green intermediate (compound 5) is not isolated by filtration.
The inventors have now developed alternative and improved methods for the synthesis of diaminophenothiazinium compounds (including, in particular, MTC). The new methods result in higher yields, have shorter reaction times and a higher throughput, require less energy input and generate less waste. The compounds obtained have high purity levels.